Reconhecemos financiar o apoio do Conselho de Pesquisa Nacional de Saúde e Medicina (conceder APP605524, 490037 e 1047082), o Australian Research Council (conceder DP12010061), a Estratégia Nacional de Infra-estrutura Collaborative Research, da Austrália e do fundo de investimento de Educação do Departamento de Inovação, Indústria , Ciência e Investigação. PML é um receptor de um prêmio de Pós-Graduação da Austrália.

Todos os procedimentos foram conduzidos de acordo com as políticas da Universidade Macquarie e conformado com o Conselho Nacional de Saúde e Pesquisa Médica (NHMRC) Código australiano de prática. O trabalho foi realizado sob as Ética Nenhum Acordo ARA 2012/017 aprovados e obtido da Comissão de Ética Animal da Universidade de Macquarie. Todas as experiências foram realizadas em ratinhos SJL / J a menos que indicado de outra forma. 1. Ratos e Experimental Malaria Infection <ol><li> Camundongos com temperatura controlada (21 ° C) com um ciclo claro-escuro de 12:12 hr casa. </li><li> Descongelar uma aliquota de criopreservado P. chabaudi Adami DS com 5% de hemácias parasitadas (PRBCs) e injetar 200 mL na cavidade intra-peritoneal de camundongos C57BL / 6 camundongo doador. </li><li> Monitorar doador rato parasitemia a cada 1-2 dias, utilizando a citometria de fluxo, conforme descrito na Seção 3-4 do presente protocolo. Uma vez que os doadores atinge 5-15% parasitemia, coleta de sangue por punção cardíaca, como segue: <ol><li>Aspirar 100 ul de solução de heparina (300 U / ml de heparina em solução de Ringer rato (MTR) (NaCl 154 mM, KCl 5,6 mM, MgCl2 1 mM, 2,2 mM de CaCl2, 20 mM de HEPES, 10 mM de glucose, 30 U / ml heparina, pH 7,4, filtro de 0,22 ^ M esterilizado)) numa seringa de 1 ml com uma agulha G 26. </li><li> Anestesiar o mouse por inalação com 5% de isoflurano, confirmando a profundidade desejada da anestesia, pela ausência de resposta de retirada pedal e dos reflexos da córnea. </li><li> Coloque rato sobre o seu lado e perpendicularmente agulha de inserção, logo abaixo do cotovelo, através das costelas, e no coração. Puxe o êmbolo da seringa lenta e gire a agulha até 0,5-1 ml de sangue é obtido. </li><li> Execute eutanásia humanitária por deslocamento cervical sob anestesia. </li></ol></li><li> Calcular as concentrado de hemácias por volume de sangue do dador assumindo uma contagem de sangue de 9 x 10 6 de RBC / ul (ou seja, se o doador estava em 10% quando a parasitemia sacrificados, o sangue conterá 9 x 10 5CH / ul). Dilui-se sangue parasitadas em solução salina de Krebs (NaCl 126 mM, KCl 2,5 mM, NaHCO 3 25, 1,2 mM de NaH 2 PO 4, 1,2 mM MgCl2, 2,5 mM de CaCl2, 0,2% de glucose, pH 7,2, filtro de 0,22 ^ M esterilizado) tamponada de modo que a concentração é de 1 x 10 4 concentrado de hemácias por 200 ul e 200 ul injectar na cavidade intraperitoneal de ratinhos necessárias para a marcação de RBC e experimento transfusão. </li><li> Monitorar parasitemia a cada 1-2 dias, utilizando a citometria de fluxo, conforme descrito nas secções 3-4 deste protocolo. </li></ol> 2. A marcação de hemácias e Transfusão <ol><li> Recolha de sangue heparinizado a partir de murganhos, por punção cardíaca, tal como descrito no passo 1.3, e colocar em gelo. Manter sangue a 4 ° C em todos os momentos. Colete aproximadamente 200 sangue ul por mouse para ser injetado. Se necessário, dividida em duas amostras de sangue e tratar uma amostra de como desejar. NOTA: Desta forma, a invasão do samp tratadale pode ser comparado com um controlo, uma amostra não tratada (ver Figura 1 para esquemática). </li><li> Prepara-se uma solução de cada etiqueta fluorescente RBC 2x. <ol><li> Dilui-se uma solução estoque a 2 mg / ml de Atto 633-N-hidroxisuccinimida (NHS Atto-633) em MTR, de modo que a concentração é de 20 ug / ml. </li><li> Dilui-se uma solução estoque de 25 mg / ml de biotina-N-hidroxissuccinimida (biotina-NHS) em MTR, de modo que a concentração é de 250 ug / ml. </li></ol></li><li> Dividir as amostras de sangue em dois tubos e adicionar um volume igual de 2x solução Atto 633-NHS para um tubo, e um volume igual de 2x a biotina-NHS para outro tubo. Misturar imediatamente. Incubar sangue a 4 ° C durante 1 h, com agitação lenta constante, ou, alternativamente, misturar cada amostra de 10 - 15 min. </li><li> Lavar 3 vezes sangue marcadas com MTRC (MTR + 0,5% albumina de soro bovino (BSA), filtro de 0,22 ^ M esterilizado) como se segue: <ol><li> Adicionar pelo menos dois volumes de MTRC, centrifugar a 750 xg durante 5 minutos, remover o sobrenadante, e resuspend o sedimento em, pelo menos, 2 volumes de MTRC. Repita mais duas vezes. </li><li> Após a lavagem final, centrifugar a 750 xg durante 5 min e combinar o sangue sedimentadas em diferentes combinações (ou seja, sem tratamento Atto 633 / Biotina tratada e não tratada Biotina / tratada Atto 633). Adicionar MTR para fazer até um volume de 200 mL por rato para ser injetado. </li></ol></li><li> Injetar sangue marcado intravascular em camundongos infectados roedor malária ou controles não infectados como se segue: <ol><li> Execute transfusão a 2 - 15% parasitemia no auge de parasita esquizogonia. NOTA: isto ocorre aproximadamente a meio do ciclo escuro (ou seja, entre 12-2 da manhã de um ciclo de luz normal, ou 12-2 pm em um ciclo de luz inverso). </li><li> Camundongos morna usando uma lâmpada de calor por 5-10 minutos para aumentar o fluxo sanguíneo, mas tomar precauções para não superaquecer os ratos. Coloque os ratos em um dispositivo de retenção e intravascular injetar 200 mL de sangue marcado (aproximadamente 1 - 2 x 10 9 RBCs) através da veia da cauda. </li></ol></li><li> Coletar amostras de sangue em diferentes momentos após a injeção, conforme descrito na Seção 3. NOTA: As taxas de invasão pode ser quantificada inferior a 10 minutos após a injecção, dependendo da parasitemia do rato destinatário. </li></ol> 3. Recolha de amostras de sangue e Preparação para citometria de fluxo <ol><li> Preparar 50 ml de solução de coloração por amostra, e um pouco mais, no dia da experiência como se segue: <ol><li> Descongelar uma aliquota de 6 JC-1 mM armazenado a -20 ° C em dimetil-sulfóxido (DMSO). </li><li> Quente 50 ul de MTRC por amostra, e um pouco mais, a 37 ° C. </li><li> Embora a vórtex continuamente MTRC pré-aquecido, adicionar JC-1, de modo que a concentração final é de 12 uM. Isto é feito para reduzir a formação de JC-1 agregados; se agregados que formam, fazer tubo não centrífuga, pois isso vai evitar manchas. NOTA: Uma pequena quantidade de agregação não afetará o resultado. </li><li> Adicionar anti-CD45 da APC eFluor 780 e anti-CD71 PerCP eFluor 710 de modo a que a concentração final é de 1 ug / ml. Se o experimento RBC marcado, adicionar estreptavidina PE-Cy7 de modo que a concentração final é de 1 ug / ml. </li></ol></li><li> Realizar cauda sangramento por amputação da ponta da cauda ou a laceração do vaso sanguíneo dentro da cauda. Coloque uma gota de sangue da cauda para um pequeno pesar barco ou lâmina de vidro. Após a coleta de sangue, garantir que o sangramento parou. Pipeta 3 ul de sangue da cauda em 50 ul de solução de coloração pré-aquecido a 37 ° C e incubar as amostras a 37 ° C durante 20 min. </li><li> Descongelar uma aliquota de 4 mM de Hoechst 33342 armazenado a -20 ° C em água destilada (se usando um laser 355 nm) ou Hoechst 34580 2 mM armazenado a -20 ° C em água destilada (se usando um laser de 405 nm). Prepare 500 mL por amostra, além de extra, de uma solução de 4 mm ou 2 M de Hoechst em MTRC, respectivamente. </li><li> Adicionar 500 uL de 4 uM Hoechst33342 ou 2 uM Hoechst 34580 para as amostras e incuba-se à temperatura ambiente durante 20 min. </li><li> Centrifugar células a 750 xg durante 3 min a 4 ° C, o sobrenadante de descarte, adicionar 700 ul MTRC e analisar por citometria de fluxo. </li></ol> 4. Citometria de Fluxo <ol><li> Analisar amostras usando um laser 355/488/633 nm ou 405/488/633 instrumento a laser nm. </li><li> Amostras de filtrar através de um filtro de células 35 mM imediatamente antes da análise. Enxágüe filtro de células com água destilada entre amostras e re-uso. </li><li> Grave eventos suficientes para que a menor população contém, pelo menos, 500 eventos (geralmente 1.000.000 - 10.000.000 Eventos totais por amostra). </li><li> Excite Hoechst 33342 usando um laser de 355 nm e detectar eventos através de um filtro 460/50. Excitar a Hoechst 34580 usando um laser de 405 nm e detectar eventos através de um filtro 460/50. Excitar JC-1, anti-CD71 PerCP eFluor 710 e PE-estreptavidina Cy7 utilizando um laser de 488 nm e detectar eventos através de um 530/40, 692/40, e filtrar 750LP respectivoly. Excite Atto 633 e anti-CD45 APC eFluor 780 usando um laser de 633 nm e detectar eventos através de um filtro de 670/30 ou 750LP respectivamente. </li><li> Realizar análise e compensação usando citometria de fluxo apropriado software de análise da seguinte forma: <ol><li> Selecione as células inteiras e não incluem ruído, resíduos, e plaquetas com base em FSC / propriedades SSC (Figura 2A). Selecione células individuais com base em qualquer largura de pulso de disparo (Figura 2B) ou usando a área do pico do FSC e à altura (Figura 2C). </li><li> Escolher a eritrócitos maduros, e excluir progenitores e leucócitos de RBC, com base na fluorescência negativa nas PerCP eFluor 710 e 780 APC eFluor canais (Figura 2D). </li><li> Se a realização do experimento RBC marcado, selecione cada população de hemácias marcadas com base em PE-Cy7 e Atto 633 fluorescência e analisá-las separadamente (Figura 3A). </li><li> Selecione concentrado de hemácias com base em fluorescência positiva para ambos JC-1 e Hoechst ( <strong&gt; Figura 2E-H). </li></ol></li></ol> 5. cálculos e estatísticas <ol><li> Calcular a parasitemia por divisão do número de concentrado de hemácias pelo número total de glóbulos vermelhos (ou seja, o número de eventos em Q2 portão dividido pelo número de eventos no portão G4). Ao realizar o ensaio de invasão de parasitemia as duas populações marcados podem ser calculadas dividindo o número de concentrado de hemácias marcadas pelo número de glóbulos vermelhos marcados em que a população (isto é, o número de eventos que são tanto em portas de L1 e Q2, dividido pelo número de eventos no portão L1). NOTA: Rotulado parasitemia varia consideravelmente dependendo de fatores como a parasitemia endógeno e número de merozoites circulantes. Por esta razão, é útil para relatar a proporção de parasitemia, que é calculado como a parasitemia da população marcada tratados dividido pelo parasitemia da população de controlo marcado em cada ratinho individual. Para corrigir possíveis efeitos da tintura, relatório resulta umaé uma relação média parasitemia nas duas combinações de corantes. </li><li> Determinar a significância estatística dos índices usando o teste t uma amostra com 1 como a média hipotética. </li></ol>

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Descrevemos um método para a medição de ambos parasitemia e merozóitos de invasão in vivo amostras. Em relação à medida da parasitemia, este método oferece uma vantagem sobre os métodos anteriores em que 10-13 HJ-GVs podem ser distinguidas a partir de concentrado de hemácias, reduzindo assim o número de eventos positivos falsos. Enquanto HJ-hemácias são geralmente rara em humanos, alguns estudos relatam altos níveis em ratos 15,16 fazendo a distinção entre essas células e...

The clinical symptoms associated with malaria occur during the Plasmodium parasite&#8217;s asexual replicative cycle within red blood cells (RBCs). Merozoites, released during the liver stage of infection, quickly attach to and invade RBCs. After gaining entry into the cell, the parasite grows and matures, eventually undergoing schizogony, splitting open the cell, and releasing a cluster of newly formed merozoites which go on to repeat this cycle. As such, an assessment of malaria infection often involves monitoring both parasitemia, which is the percentage of RBCs appropriated by one or more parasites, and the rate of merozoite invasion into uninfected RBCs. 
Flow cytometry is a powerful tool which can be used to record the properties of vast numbers of cells in a short period of time. This technique has clear applicability for the measurement of malaria parasitemia and invasion, and offers several advantages over traditional microscopy techniques. These include the accurate measurement of very low parasitemia, which would be prohibitively time consuming by microscopy, the unbiased nature of the measurement, and the ability to measure multiple cell parameters simultaneously. Flow cytometry is widely used to determine both parasitemia and merozoite invasion in in vitro culture1-9, however, techniques for measuring these parameters in vivo are less well developed, and can be complicated by the presence of additional cell types which interfere with analysis. No assays have been described for measurement of in vivo invasion, and while some assays exist for the analysis of in vivo parasitemia, these lack the ability to distinguish between parasitized RBCs (pRBCs) and RBCs containing Howell-Jolly bodies (HJ-RBCs)10-13. The later issue is particularly important as in mice HJ-RBCs may account for up to 0.9% of mature RBCs14-16, thereby preventing the accurate measurement of low parasitemia. 
We have previously demonstrated an approach for the measurement of parasitemia and merozoite invasion in a rodent model of malaria infection14. Here, we provide a more detailed protocol and accompanying video. This approach builds on previous methodologies and allows for the accurate identification of parasitized RBCs, as distinct from leukocytes, RBC progenitors, and HJ-RBCs. Additionally, this assay allows the simultaneous measurement of merozoite invasion into two labeled RBC populations, a treated, or target, population, and a control population, thereby providing a robust platform for the assessment of invasion into different cell types.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>bisBenzimide H 33342 trihydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>B2261</td>
			<td>Hoechst 33342. Store a 4mM stock solution at -20 &#176;C in distilled water</td>
		</tr>
		<tr>
			<td>Hoechst 34580</td>
			<td>Sigma-Aldrich</td>
			<td>63493</td>
			<td>Store a 2mM stock solution at -20 &#176;C in distilled water</td>
		</tr>
		<tr>
			<td>JC-1 Dye</td>
			<td>Life Technologies</td>
			<td>T-3168</td>
			<td>Store small aliquots of 6mM stock solution at -20 &#176;C in DMSO</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD45 APC-eFluor 780</td>
			<td>eBioscience</td>
			<td>47-0451-80</td>
			<td>Clone 30-F11</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD71 PerCP-eFluor 710</td>
			<td>eBioscience</td>
			<td>46-0711-80</td>
			<td>Clone R17217</td>
		</tr>
		<tr>
			<td>Atto 633 NHS ester</td>
			<td>Sigma-Aldrich</td>
			<td>1464</td>
			<td>Atto 633-NHS. Store a 2mg/ml stock solution at -20 &#176;C in DMF</td>
		</tr>
		<tr>
			<td>EZ-Link Sulfo-NHS-LC-Biotin</td>
			<td>Thermo Fisher Scientific</td>
			<td>21335</td>
			<td>Biotin-NHS. Store a 25mg/ml stock solution at -20 &#176;C in DMF</td>
		</tr>
		<tr>
			<td>Streptavidin PE-Cyanine7</td>
			<td>eBioscience</td>
			<td>25-4317-82</td>
			<td>Streptavidin PE-Cy7</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H478</td>
			<td></td>
		</tr>
		<tr>
			<td>35&#181;M filter cap tubes</td>
			<td>Becton Dickinson</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer: BD LSRFortessa</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer: BD FACSAria II</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer: BD Influx</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer: CyAn ADP Analyzer</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo assessment of rodent plasmodium parasitemia and merozoite invasion by flow cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

Medição da parasitemia. Para a medição da parasitemia, células sanguíneas devem primeiro ser seleccionado, e o ruído, detritos e plaquetas excluídos, com base no FSC / SSC propriedades (Figura 2A). Dependendo do citómetro usado, células individuais devem, então, ser seleccionado com base em qualquer largura de impulsos de disparo (Figura 2B), ou FSC altura de pico com razão de área (Figura 2C). Restante eventos deve ser composto de...

Watch this Scientific Journal Video about In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry at JoVE.com

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

Os invade parasita da malária e repetições dentro das células vermelhas do sangue. A avaliação precisa da invasão merozoite e parasitemia é fundamental para avaliar o curso da infecção da malária. Aqui nós descrevemos um protocolo de citometria de fluxo com base para a medição desses parâmetros em um modelo de rato da malária.

A avaliação in vivo de Rodent Plasmodium parasitemia e Merozoito Invasion por citometria de fluxo

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

in-vivo-assessment-rodent-plasmodium-parasitemia-merozoite-invasion

Research

JoVE Journal

Immunology and Infection

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

9.5K visualizações.  Macquarie University.  Os invade parasita da malária e repetições dentro das células vermelhas do sangue. A avaliação precisa da invasão merozoite e parasitemia é fundamental para avaliar o curso da infecção da malária. Aqui nós descrevemos um protocolo de citometria de fluxo com base para a medição desses parâmetros em um modelo de rato da malária. 

Vídeo: In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Medir a atividade das calpaínas em células fixas e Vivendo por citometria de fluxo

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Imunologia e Infecção

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

Avaliação quantitativa das células imunes no tecido medula lesionada por Citometria de Fluxo: um novo uso para um método de purificação celular

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Humano In Vitro Supressão como ferramenta de triagem para o reconhecimento de um Estado precoce de desequilíbrio imunológico

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

Exame de seleção positiva e negativa Tímico por citometria de fluxo

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

Caracterização fenotípica dos macrófagos a partir de rim de rato por Citometria de Fluxo

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Avaliando Retinal microglia fagocítica Função<em&gt; In Vivo</em&gt; Usando um ensaio baseado em citometria de fluxo

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Citometria de fluxo de partículas ligadas aos Bet v 1 alérgeno no PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Isolamento e cultura In Vitro de macrófagos alveolares murino e humanos

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Caracterização de subconjuntos de monócitos humanos por sangue total Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

Discriminação de sete subconjuntos de células imunes por dois-fluorocromo citometria de fluxo

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...